[ProteinLoverInTraining]

Hi,
 
 I'm an undergraduate student currently doing a one year research placement as part of my degree.
 
 According to recent literature, a cytokine I'm working with is cleaved into a more active form by at least two serine proteases, but cleaved and inactivated by a cysteine protease. In order to obtain a native source of the cytokine, I culture a human cell line and then lyse the cells using multiple freeze-thawing cycles (the cells do not secrete the cytokine in question). I've found that adding a universal protease inhibitor cocktail to the culture during the freeze-thawing process greatly reduces cleavage of the cytokine (shown by IP) and its activity (measured using a reliable and well established reporter gene assay).
 
 Now I would like to lyse the cells in the presence of inhibitors which only inhibit serine proteases, but I am unsure which ones to use, and how likely they are to inhibit the serine proteases I'm interested in (I can't find any specific inhibitors for these) while at the same time having minimal effects on the activity of other classes of proteases, particularly cytseine proteases.

[Yggdrasil]

PMSF (phenylmethylsulfonyl fluoride), AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride), and benzamidine are all small molecules commonly used to broadly inhibit serine proteases (although they may not inhibit all, for example, benzamidine works best against the trypsin-like enzymes).  These inhibitors, however, should not affect cysteine proteases.

If you know the identities of the two serine proteases that act against your cytokine, you can search the literature to see whether these compounds will inhibit them (or you can try to figure out which class of serine protease they are most related to and use that information to guide your choice).  Of course, since you have a nice experimental readout of proteases activity, you could also just perform an experiment to see whether they inhibit cleavage of your cytokine.

[Babcock_Hall]

Stay away from leupeptin and E-64.  Some sources of information say that PMSF inhibits papain which is a cysteine protease, as well as serine proteases.  There may be a work-around to this problem, however, in that DTT is said to reverse this inhibition.
http://www.piercenet.com/browse.cfm?fldID=02040802
http://wolfson.huji.ac.il/purification/PDF/Protease_Inhibitors/ProteaseInhibitorRoche.pdf